Abstract
Single-molecule-based super-resolution microscopy offers a unique opportunity for quantifying protein copy-number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here, we show that DNA origami in combination with GFP antibodies is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy-number in cellular contexts using super-resolution microscopy.
Highlights
Single-molecule-based super-resolution microscopy offers a unique opportunity for quantifying protein copy-number with nanoscale resolution
Recent work focused on developing analytical approaches and calibration standards aimed to overcome this challenge[2,3,4,5,6,7,8,9,10] in particular to calibrate and count photoactivatable fluorescent proteins (FPs)[2,3,5,8,9]
Due to their high photon budget compared to FPs, small organic fluorophores are popular probes for many super-resolution studies
Summary
Single-molecule-based super-resolution microscopy offers a unique opportunity for quantifying protein copy-number with nanoscale resolution. The handles projecting out from the chassis provide site- and sequence-specific attachment points for single fluorophores as well as proteins of interest and allow testing of several labeling strategies (Figure 1a).
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